Yang Han N, Park Ji S, Na Kun, Woo Dae G, Kwon Young D, Park Keun-Hong
Department of Biomedical Science, College of Life Science, CHA University, 606-16 Yeoksam 1-dong, Kangnam-gu, Seoul 135-081, Republic of Korea.
Biomaterials. 2009 Oct;30(31):6374-85. doi: 10.1016/j.biomaterials.2009.07.062. Epub 2009 Aug 13.
This study was conducted to reveal the chondrogenesis of mesenchymal stem cells that had been genetically modified with the green fluorescence protein (GFP) gene and then co-cultured with chondrocytes in vitro and in vivo. Subsequent mixing of chondrocytes in the hydrogel constructs induced increased chondrogenic differentiation of the transfected hMSCs. The proliferation and differentiation of MSCs that were transfected with the GFP gene and co-cultured with chondrocytes (1:1 and 1:3) or chondrocytes alone were evaluated by a live/dead assay, MTT assay, GAG & DNA assay, RT-PCR, real time-PCR, and histological and immunochemical analysis in vitro and in vivo. Real-time PCR revealed that the expression of aggrecan and COMP by genetically modified hMSCs co-cultured with chondrocytes was 2 or 3 times greater than that of genetically modified MSCs alone. Moreover, the expression of collagen type II was more than 3.5 times greater than that of genetically modified MSCs alone. 3-D hydrogel constructs co-cultured with chondrocytes and genetically modified MSCs showed a significantly higher number of specific lacunae phenotypes at the end of the 4 week study, regardless of whether they were co-cultured in the presence of chondrocytes. These findings indicate that co-culture with chondrocytes and genetically modified MSCs can be used to engineer well designed implants for the formation of neocartilage by transplanted genetically modified MSCs.
本研究旨在揭示经绿色荧光蛋白（GFP）基因修饰的间充质干细胞在体外和体内与软骨细胞共培养后的软骨形成情况。随后在水凝胶构建物中混合软骨细胞可诱导转染的人骨髓间充质干细胞（hMSCs）软骨分化增加。通过活/死检测、MTT检测、糖胺聚糖（GAG）与DNA检测、逆转录聚合酶链反应（RT-PCR）、实时定量PCR以及体外和体内的组织学和免疫化学分析，评估了转染GFP基因并与软骨细胞（1:1和1:3）或单独与软骨细胞共培养的间充质干细胞的增殖和分化情况。实时PCR显示，与软骨细胞共培养的基因修饰hMSCs中聚集蛋白聚糖和软骨寡聚基质蛋白（COMP）的表达比单独的基因修饰间充质干细胞高2至3倍。此外，II型胶原蛋白的表达比单独的基因修饰间充质干细胞高3.5倍以上。在为期4周的研究结束时，与软骨细胞和基因修饰间充质干细胞共培养的三维水凝胶构建物显示出显著更多的特定陷窝表型，无论它们是否在软骨细胞存在下共培养。这些发现表明，与软骨细胞和基因修饰间充质干细胞共培养可用于设计良好的植入物，通过移植基因修饰的间充质干细胞来形成新软骨。
